GoKawiilCell culture
A375, HEK293T, Sk-Mel-3 and Sk-Mel-24 cell lines were obtained from the American Type Culture Collection. A375 and HEK293T cells were maintained in high-glucose Dulbecco’s modified Eagle’s medium supplemented with L-glutamine, sodium pyruvate (Gibco; 11995065) and 10% fetal bovine serum (FBS) (Sigma-Aldrich; F4135). Sk-Mel-3 cells were maintained in McCoy’s 5A medium (Gibco; 16600082) supplemented with 10% FBS, and Sk-Mel-24 cells were maintained in Eagle’s minimum essential medium (American Type Culture Collection; 30-2003) supplemented with 15% FBS. All cells were cultured at 37 °C with 5% CO 2 .
Cell strain construction
To preserve the potential clonal diversity of cancer cells, we adopted a simplified engineering strategy for the A375 cell line, rather than transducing Tet-On dCas9–KRAB–MeCP2 followed by monoclonal selection, as in our previous study15. Inspired by the Genome-Wide Perturb-Seq study8, we selected dCas9–BFP–KRAB as the CRISPRi effector. The A375–dCas9–BFP–KRAB cell strain was generated by transducing A375 cells with lentivirus produced from pHR–SFFV–dCas9–BFP (Addgene; 46910) at MOI of 0.2. BFP+ cells within the top 15% of fluorescence intensity were sorted and expanded. Enrichment sorting was performed regularly to maintain Cas9 expression levels in the cell strain.
Plasmid and lentivirus construction
Single sgRNA expression construct cloning and lentivirus packaging were performed as in our previous study15. In brief, the CROPseqOPTI vector with an additional EGFP marker (CROPseqOPTI–GFP) was digested with BsmBI (New England Biolabs; ER0451) to remove the filler sequence between the U6 promoter and the sgRNA scaffold. Pre-annealed phosphorylated double-stranded DNA with an sgRNA sequence and complementary sticky ends was ligated into the digested backbone. Lentivirus was packaged by co-transfecting the sgRNA-expressing plasmid, psPAX2 plasmid and pMD2.G plasmid into HEK293T cells at high confluency. After washing the cells and changing the medium, virus-containing supernatants were collected 24 h later. The functional titre of the viruses was measured by assessing the proportion of fluorescent-marker-positive cells after transducing target cell lines with varying amounts of lentivirus.
A two-step cloning strategy was adopted for the construction of our dual-sgRNA drug resistance library. The oligo library was designed with the following structure: BsmBI digestion site–sgRNA1 sequence–PaqCI digestion site–filler–PaqCI digestion site–sgRNA2 sequence–BsmBI digestion site. The single-stranded library (Integrated DNA Technologies) was amplified into double-stranded DNA using primers complementary to both ends and then digested with BsmBI. The amplified library with sticky ends was ligated into the BsmBI-digested CROPseqOPTI–GFP backbone using Blunt/TA Ligase Master Mix (New England Biolabs; M0367S). The ligation product was purified with 0.75× AMPure beads (Beckman Coulter; A63881) and transformed into competent cells through electroporation. After recovery, cells were spread onto a bioassay agarose plate and incubated at 32 °C for 14 h. A separate agarose plate at a 1:100,000 dilution was inoculated to determine sgRNA coverage. All colonies were scrapped, and a Midiprep was performed to obtain the intermediate plasmid library.
The intermediate plasmid library and a gBlock (Integrated DNA Technologies) with the structure ‘PaqCI digestion site–sgRNA scaffold CR3 with a 10× capture sequence–H1 promoter–PaqCI digestion site’ were digested with PaqCI (New England Biolabs; R0745S). Ligation, transformation and plasmid preparation were performed as described above.
The full structure of the sgRNA expression cassette in the final library is U6 promoter–sgRNA1 sequence–sgRNA scaffold CR3 with a 10× capture sequence–H1 promoter–sgRNA2 sequence–sgRNA scaffold. The accuracy of plasmid library construction and the abundance of each sgRNA pair were evaluated using amplicon sequencing.
Dual-sgRNA-expressing CROPseqOPTI–mCherry plasmids were constructed for specific gene knockdown experiments. In brief, gBlocks with the structure ‘homology arm to the U6 promoter sequence–sgRNA1 sequence–sgRNA scaffold CR3 with a 10× capture sequence–H1 promoter–sgRNA2 sequence–homology arm to the second sgRNA scaffold’ were mixed with the BsmBI-digested CROPseqOPTI–mCherry backbone for Gibson assembly. After incubation at 50 °C for 30 min, transformation and plasmid preparation were carried out as described in our previous study15.
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